EP2129394B1 - Impfstoffzusammensetzung gegen papillomavirus - Google Patents

Impfstoffzusammensetzung gegen papillomavirus Download PDF

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EP2129394B1
EP2129394B1 EP08726508A EP08726508A EP2129394B1 EP 2129394 B1 EP2129394 B1 EP 2129394B1 EP 08726508 A EP08726508 A EP 08726508A EP 08726508 A EP08726508 A EP 08726508A EP 2129394 B1 EP2129394 B1 EP 2129394B1
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hpv
vlps
adjuvant
aluminum
type
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EP2129394A1 (de
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Janine T. Bryan
Michelle K. Brownlow
Li Shi
Danilo Casimiro
William L. Mcclements
Brian K. Meyer
Binghua Hu
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Merck Sharp and Dohme LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates generally to the prevention of human papillomavirus (HPV) infection. More specifically, the invention relates to pharmaceutical compositions and formulations comprising virus-like particles (VLPs) of HPV, said VLPs adsorbed to an aluminum adjuvant, and a second adjuvant comprising a saponin, cholesterol, and a phospholipid. The invention also relates to pharmaceutical compositions and formulations comprising HPV VLPs and an antimicrobial preservative. Further provided are methods of using the disclosed pharmaceutical composition and formulations.
  • VLPs virus-like particles
  • HPV human papillomavirus
  • HPV types 6 and 11 are termed "low-risk" and are the HPV types which are most commonly associated with benign warts, nonmalignant condyloma acuminata and/or low-grade dysplasia of the genital or respiratory mucosa. Approximately 90% of genital warts are caused by these two HPV types.
  • HPV 16 and HPV 18 are termed "high-risk" HPV types because they are most frequently associated with in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal. More than 70% of cervical carcinomas are caused by infections with HPV 16 and HPV 18. Together with the less prevalent oncogenic types HPV 31, -33, -45, - 52 and -58, these types account for greater than 90% of cervical cancer ( Schiffman et al., J Natl. Cancer Inst. 85(12): 958-64 (1993 )). Cervical cancer is the second most prevalent cause of cancer deaths in women worldwide.
  • Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode up to eight early (E1- E7) and two late (L1-L2) genes.
  • the L1 protein is the major capsid protein and has a molecular weight of 55-60 kDa.
  • VLPs virus-like particles
  • VLPs are morphologically similar to authentic virions and are capable of inducing high titres of neutralizing antibodies upon administration into animals or humans. Because VLPs do not contain the potentially oncogenic viral genome, they present a safe alternative to the use of live virus in HPV vaccine development (for review, see Schiller and Hidesheim, J. Clin. Virol. 19: 67-74 (2000 )). For this reason, the L1 and L2 genes have been identified as immunological targets for the development of prophylactic and therapeutic vaccines for HPV infection and disease.
  • VLP-based vaccines have proven to be effective at inducing immune responses in human patients vaccinated with bivalent HPV 16 and 18 ( Harper et al. Lancet 364(9447): 1757-65 (2004 )) and quadrivalent HPV 6, 11, 16, and 18 VLP-based vaccines ( Villa et al. Vaccine 24: 5571-5583 (2006 )). However, it is a common goal of vaccine development to augment the immune response to the desired antigen to induce long lasting protective immunity.
  • adjuvants Co-administration of vaccines with compounds that can enhance the immune response against the antigen of interest, known as adjuvants, has been extensively studied. In addition to increasing the immune response against the antigen of interest, some adjuvants may be used to decrease the amount of antigen necessary to provoke the desired immune response or decrease the number of injections needed in a clinical regimen to induce a durable immune response and provide protection from disease.
  • Aluminum-based compounds were determined to possess adjuvant activity over 60 years ago (for review, see Lindblad, E.B. Immunol. and Cell Biol. 82: 497-505 (2004 ); Baylor et al. Vaccine 20: S18-S23 (2002 )). Aluminum adjuvants are generally regarded as safe when used at appropriate dosages. Many have been approved for administration into humans by regulatory agencies worldwide.
  • the present invention relates to pharmaceutical compositions and formulations that can induce an immune response against HPV in a patient.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising, in each dose: (a) from 10 to 100 ⁇ g per type of virus-like particles (VLPs) of at least one type of human papillomavirus (HPV); (b) from 300 ⁇ g to 1200 ⁇ g/ml of an aluminum adjuvant, (c) from 20-400 ⁇ g/ml of an ISCOM type adjuvant which comprises an immune-stimulating complex comprised of a saponin, cholesterol and a phospholipid, and (d) a pharmaceutically acceptable carrier, wherein said VLPs are comprised of recombinant L1 protein or recombinant L1+L2 proteins of HPV, and wherein said VLPs are adsorbed to said aluminum adjuvant.
  • VLPs virus-like particles
  • HPV human papillomavirus
  • the pharmaceutically acceptable carrier may contain a buffer, normal saline or phosphate buffered saline, sucrose, other salts and/or polysorbate.
  • the HPV type is selected from the group consistin of HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV39, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV58, HPV59. HPV66, HPV68, HPV73 and HPV82.
  • the composition comprises VLPs of HPV types 6, 11, 16, 18.
  • the composition comprises VLPs of types 31, 45, 52, 58.
  • composition may further comprise VLPs of an additional HPV type selected from the group consisting of: HPV26, HPV33, HPV35, HPV39, HPV51, HPV53, HPV55, HPV56, HPV59, HPV66, HPV68, HPV73, HPV82.
  • VLPs of an additional HPV type selected from the group consisting of: HPV26, HPV33, HPV35, HPV39, HPV51, HPV53, HPV55, HPV56, HPV59, HPV66, HPV68, HPV73, HPV82.
  • the composition comprises, in each dose: (a) up to 600 ⁇ g total VLPs; (b) from 150 ⁇ g to 600 ⁇ g of aluminum adjuvant; (c) from 10 ⁇ g to 200 ⁇ g of ISCOM-type adjuvant.
  • the composition comprises, in each dose: (a) from 20 ⁇ g to 80 ⁇ g VLPs of any one HPV type; (b) from 200 ⁇ g to 300 ⁇ g of aluminum adjuvant; (c) from 15 ⁇ g to 120 ⁇ g of ISCOM-type adjuvant.
  • the composition comprises, in each dose: (a) from 20 ⁇ g to 80 ⁇ g VLPs of any one HPV type; (b) from 300 ⁇ g to 500 ⁇ g of aluminum adjuvant; (c) from 15 ⁇ g to 120 ⁇ g of ISCOM-type adjuvant.
  • the composition comprises, in each dose: (a) from 20 to 80 ⁇ g of VLPs of each HPV types 6, 11, 16, 18, 31, 45, 52 and 58, said HPV VLPs being comprised of recombinant L1 or recombinant L1+L2 proteins of HPV; (b) from 200 ⁇ g to 500 ug of aluminum adjuvant; and (c) from 30 ⁇ g to 120 ⁇ g of an ISCOM-type adjuvant, wherein said HPV are adsorbed onto said aluminium adjuvant.
  • the composition further comprises an antimicrobial preservative selected from the group consisting of m-cresol, phenol, and benzyl alcohol.
  • the antimicrobial preservative consists of m-cresol at a concentration of 0.15% to 0.31 %, phenol at a concentration of 0.25% to 0.55%, or benzyl alcohol at a concentration of 0.75% to 1.2%.
  • the aluminum adjuvant is selected from the group consisting of aluminum hydroxyphosphate (AlPO 4 ), amorphous aluminum hydroxyphosphate sulphate (AAHS), and aluminum hydroxide (Al(OH) 3 ).
  • AlPO 4 aluminum hydroxyphosphate
  • AAHS amorphous aluminum hydroxyphosphate sulphate
  • Al(OH) 3 aluminum hydroxide
  • the aluminum adjuvant comprises or AlPO 4 AAHS, and phosphate and aluminum are present in a molar ratio of 0.1 to 1.1 phosphate to aluminum. In another embodiment, this molar ratio is of 0.2 to 0.5.
  • the ISCOM-type adjuvant is ISCOMATRIX®.
  • HPV vaccine formulations comprising HPV VLPs of at least one HPV type, an ISCOM-type adjuvant and an aluminum adjuvant are able to provoke higher Ab titers to the targeted HPV types in animal models and in human patients when compared to VLP vaccines comprising aluminum adjuvant alone.
  • HPV VLP vaccine formulations and pharmaceutical compositions are provided herein that comprise HPV VLPs which are adsorbed onto aluminum adjuvant in combination with an ISCOM-type adjuvant, as described in detail below.
  • HPV vaccine formulations and pharmaceutical compositions comprising HPV VLPs and an antimicrobial preservative selected from the group consisting of: m-cresol, phenol and benzyl alcohol are provided.
  • Such vaccine compositions may also include and ISCOM-type adjuvant and an aluminum adjuvant, as described above.
  • Virus-like particle-based vaccines have proven to be safe and effective at inducing immune responses against many types of HPV in human patients.
  • VLPs can self-assemble when L1, the major capsid protein of human and animal papillomaviruses, is expressed in yeast, insect cells, mammalian cells or bacteria (for review, see Schiller and Roden, in Papillomavirus Reviews: Current Research on Papillomaviruses; Lacey, ed. Leeds, UK: Leeds Medical Information, pp 101-12 (1996 )).
  • Morphologically indistinct HPV VLPs can also be produced by expressing a combination of the L1 and L2 capsid proteins.
  • VLPs are morphologically similar to authentic virions and are capable of inducing high titres of neutralizing antibodies upon administration into an animal. Immunization of rabbits ( Breitburd et al., J. Virol. 69(6): 3959-63 (1995 )) and dogs ( Suzich et al., Proc. Natl. Acad. Sci. USA 92(25): 11553-57 (1995 )) with VLPs was shown to both induce neutralizing antibodies and protect against experimental papillomavirus infection. Additionally, immunization of adult women with HPV 16 VLPs was shown to protect against HPV 16 infection and HPV 16 cervical intraepithelial neoplasia ( Koutsky et al. N. Engl. J.
  • VLPs do not contain the potentially oncogenic viral genome and can self-assemble when expressed from a single gene, they present a safe alternative to the use of live virus in HPV vaccine development (for review, see Schiller and Hidesheim, J. Clin. Virol. 19: 67-74 (2000 )).
  • VLP-based vaccines have proven to be effective at inducing immune responses in many animal models and in human patients.
  • a goal of vaccine development is often to induce higher antibody titers, leading to long lasting protective immunity.
  • pharmaceutical compositions and vaccines comprising HPV VLPs of at least one HPV type, an ISCOM-type adjuvant and an aluminum adjuvant have been developed, as disclosed herein.
  • Said pharmaceutical compositions and vaccines are able to provoke higher IgG Ab titers to the targeted HPV types than VLP vaccines comprising aluminum adjuvant alone. This combination can lead to the induction of long-lasting effective immunoprophylaxis against HPV infection, reduced antigen dosage, and immunization schedules comprising a decreased number of injections over time.
  • the present invention relates to pharmaceutical compositions and formulations comprising HPV virus-like particles, an aluminum adjuvant, an ISCOM-type adjuvant, and a pharmaceutically acceptable carrier, wherein said VLPs are comprised of recombinant L1 protein or recombinant L1 + L2 proteins of HPV and wherein said VLPs are adsorbed to said aluminum adjuvant.
  • HPV VLP-based vaccine is suitable for use in the pharmaceutical compositions of the present invention.
  • Known HPV VLP vaccines can be modified to include both an aluminum adjuvant and an ISCOM-type adjuvant.
  • new vaccines can be developed according to the invention described herein that comprise at least one HPV type in the form of an HPV VLP adsorbed to an aluminum adjuvant in combination with an ISCOM-type adjuvant.
  • An exemplary HPV VLP vaccine is the Quadrivalent Human Papillomavirus (Types 6, 11, 16, 18) Recombinant Vaccine, which is referred to herein by its proprietary name GARDASIL® (see Bryan, J.T. Vaccine 25(16): 3001-6 (2007 ); Shi et al. Clinical Pharmacology and Therapeutics 81 (2): 259-64 (2007 )).
  • GARDASIL® is a non-infectious recombinant, quadrivalent vaccine prepared from highly purified VLPs of the major capsid (L1) protein of HPV types 6, 11, 16, and 18. The L1 proteins are produced by separate fermentations in recombinant Saccharomyces cerevisiae and self-assembled into VLPs.
  • each GARDASIL® vaccine dose contains aluminum adjuvant (as amorphous aluminum hydroxyphosphate sulfate), sodium chloride, L-histidine, polysorbate 80, sodium borate, and water. Therefore, the present invention includes the combination of GARDASIL® with an ISCOM-type adjuvant, such as ISCOMATRIX® (CSL Ltd., Parkville, Australia).
  • the pharmaceutical compositions and formulations comprise HPV VLP-based vaccines which are monovalent, bivalent, or trivalent.
  • HPV VLP-based vaccines which are monovalent, bivalent, or trivalent.
  • pharmaceutical compositions comprising VLPs of HPV 16 and/or HPV 18, without the inclusion of other HPV VLP types, are included within the scope of the invention.
  • Quadrivalent vaccines comprising different HPV VLPs than the HPV types included in GARDASIL® are also contemplated herein.
  • pharmaceutical compositions comprising VLPs of HPV types 31, 45, 52, and 58 are within the scope of this invention.
  • the pharmaceutical compositions comprise VLP-based vaccines with more than four different types of VLPs.
  • the pharmaceutical composition comprises eight different HPV VLP types.
  • a particularly preferred octavalent vaccine is described in EXAMPLES 1 and 2 and comprises HPV types 6, 11, 16, 18, 31, 45, 52, and 58.
  • An alternative octavalent composition of the present invention comprises HPV types 6, 11, 16, 18, 31, 35, 45, and 58.
  • Other octavalent HPV VLP vaccines are also contemplated herein; for example, HPV 33 may be substituted for HPV 31.
  • Additional HPV VLPs may also be added to the vaccine formulations described herein, leading to HPV vaccines that are 9-valent, 10-valent, and so forth.
  • the pharmaceutical compositions and formulations of the present invention comprise at least one HPV VLP type, such as HPV 16 or 18.
  • the vaccine further comprises VLPs of at least one additional HPV type.
  • the at least one additional HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV 18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82.
  • compositions of the present invention comprise HPV VLPs comprised of recombinant L1 or recombinant L1 + L2 proteins of HPV.
  • HPV L1 or L1 + L2 protein can be expressed recombinantly by molecular cloning of L1 or L1 + L2 DNA into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant protein. Techniques for such manipulations are fully described by Sambrook et al. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989 )), which is hereby incorporated by reference. VLPs can self-assemble when L1 protein is recombinantly expressed in a host cell.
  • the recombinant HPV L1 proteins of the present invention may be any full-length L1 protein sequence that can be found in nature or any mutated or truncated L1 protein that is capable of self-assembling into VLPs.
  • L1 protein sequences for use in the present invention can be determined by isolating DNA from one or more clinical samples containing an HPV type of choice, determining the sequence of the HPV L1 DNA sequence, and translating the DNA sequence into an amino acid sequence using the genetic code.
  • Many exemplary L1 sequences suitable for use in the present invention can be found in the literature. See, e.g., U.S. Patent Nos.
  • L1 proteins that are useful in the compositions and formulations of the present invention include biologically active fragments and/or mutants of an HPV L1 sequence, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations, such that these mutations provide for L1 proteins or protein fragments that are capable of forming a VLP. See, e.g., International Publication WO 2006/114312 and US Patent No. 6,599,508 .
  • Appropriate host cells for the expression of recombinant HPV L1 or recombinant L1 + L2 and subsequent self-assembly of VLPs include, but are not limited to yeast cells, insect cells, mammalian cells or bacteria.
  • the VLPs are produced in yeast cells such as a yeast selected from the group consisting of: Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Kluyvermyces fragilis, Kluveromyces lactis, and Schizosaccharomyces pombe. Expression of HPV VLPs in yeast cells offers the advantages of being cost-effective and easily adapted to large-scale growth in fermenters.
  • the present invention also includes pharmaceutical compositions comprising mutant forms of HPV VLPs, such as HPV VLPs that comprise biologically active fragments and/or mutants of an HPV L 1 or L2 protein, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of therapeutic or prophylactic use and would be useful for HPV VLP vaccine development.
  • Any such mutant form of an HPV L1 protein should be capable of forming VLPs and of provoking an immune response against the desired HPV type when administered to a human.
  • L1 or L1 + L2 protein which is used to-self-assemble VLPs for inclusion in the compositions disclosed herein, may be encoded by a full-length wild-type HPV L1 or L2 polynucleotide, or may be encoded by a fragment or mutant of the known wild-type sequence. Wild-type polynucleotide sequences that encode mRNA expressing HPV L1 or L2 protein are available in the art.
  • Any mutant polynucleotide will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of an HPV L1 or L2 protein, including the ability to form VLPs that are able to provoke an immune response against the HPV type of interest when administered to a human.
  • Any such polynucleotide includes but is not necessarily limited to: nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations.
  • An immunologically or prophylactically effective dose comprises 10 ⁇ g to 100 ⁇ g, and preferably about 20 ⁇ g to 80 ⁇ g of VLPs.
  • Aluminum has long been shown to stimulate the immune response against co-administered antigens, primarily by stimulating a T H 2 response.
  • the formulations of this aspect of the present invention are adsorbed to aluminum adjuvant. It is preferred that the aluminum adjuvant of the compositions provided herein is not in the form of an aluminum precipitate.
  • Aluminum-precipitated vaccines may increase the immune response to a target antigen, but have been shown to be highly heterogeneous preparations and have had inconsistent results ( see Lindblad E.B. Immunology and Cell Biology 82: 497-505 (2004 )).
  • Aluminum-adsorbed vaccines in contrast, can be preformed in a standardized manner, which is an essential characteristic of vaccine preparations for administration into humans. Moreover, it is thought that physical adsorption of a desired antigen onto the aluminum adjuvant has an important role in adjuvant function, perhaps in part by allowing a slower clearing from the injection site or by allowing a more efficient uptake of antigen by antigen presenting cells.
  • the aluminum adjuvant of the present invention may be in the form of aluminum hydroxide (Al(OH) 3 ), aluminum phosphate (AlPO 4 ), aluminum hydroxyphosphate, amorphous aluminum hydroxyphosphate sulfate (AAHS) or so-called "alum" (KAl(SO 4 ) ⁇ 12H 2 O) ( see Klein et al., Analysis of aluminum hydroxyphosphate vaccine adjuvants by (27)A1 MAS NMR., J Pharm. Sci. 89(3): 311-21 (2000 )).
  • the aluminum adjuvant is aluminum hydroxyphosphate or AAHS.
  • the ratio of phosphate to aluminum in the aluminum adjuvant can range from 0 to 1.3. In preferred embodiments of this aspect of the invention, the phosphate to aluminum ratio is within the range of 0.1 to 0.70. In particularly preferred embodiments, the phosphate to aluminum ratio is within the range of 0.2 to 0.50.
  • the aluminum adjuvant is in the form of AAHS (referred to interchangeably herein as Merck aluminum adjuvant (MAA)).
  • MAA carries zero charge at neutral pH, while AlOH carries a net positive charge and AlPO 4 typically carries a net negative charge at neutral pH.
  • MAA has a higher capacity to bind HPV VLPs than AlOH.
  • VLPs adsorbed to MAA can induce a greater humoral immune response in mice than VLPs adsorbed to AlOH. Caulfield et al., Human Vaccines 3: 139-146 (2007 ).
  • the aluminum adjuvant of the pharmaceutical compositions of the present invention have zero point surface charge at neutral pH.
  • One of skill in the art will be able to vary the buffer, salt concentration and/or percent of free phosphate in order to allow a zero point surface charge at neutral pH.
  • An effective and safe dose of aluminum adjuvant varies from 150 to 600 ⁇ g/dose (300 to 1200 ⁇ g/mL concentration). In specific embodiments of the formulations and compositions of the present invention, there is between 200 and 300 ⁇ g aluminum adjuvant per dose of vaccine. In alternative embodiments of the formulations and compositions of the present invention, there is between 300 and 500 ⁇ g aluminum adjuvant per dose of vaccine.
  • one aspect of the present invention provides vaccines and formulations which comprise HPV VLPs in combination with an aluminum adjuvant and an ISCOM-type adjuvant.
  • the ISCOM-type adjuvant comprises a saponin, cholesterol, and a phospholipid, and forms an immune-stimulating complex or ISCOM.
  • the potent adjuvant activity of saponins which are typically isolated from the bark of the Quillaia saponaria tree, was first documented over 80 years ago (for review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25 (1996 ); and Skene and Sutton, Methods 40: 53-59 (2006 )).
  • ISCOM-type adjuvants or ISCOMs are able to provoke a broader immune response to a co-administered antigen, comprising both T-cell and antibody responses.
  • a potential for toxicity and haemolytic activity was found, limiting the promise of saponins for human or animal use at that time.
  • saponins when combined with cholesterol and phospholipid, form a characteristic particle having a caged-like structure comprised of twenty or more subunits. This unique structure contributes to the adjuvant activity of the ISCOMs. Additionally, the incorporation of saponins into ISCOMs, together with cholesterol and phospholipid, was shown to eliminate the haemolytic activity of saponins. It was also shown that less adjuvant was needed to induce an immune response when ISCOMs were utilized as adjuvant compared to free saponins ( see Skene and Sutton, supra ). For these reasons, ISCOMs have been intensely studied as potential vaccine adjuvants.
  • the present invention relates to pharmaceutical compositions comprising VLPs of at least one type of HPV, an aluminum adjuvant, an ISCOM-type adjuvant, and a pharmaceutically acceptable carrier, said ISCOM-type adjuvant comprising a saponin, cholesterol, and a phospholipid, wherein said VLPs are comprised of recombinant L1 protein or recombinant L1 + L2 proteins of HPV and wherein said VLPs are adsorbed to said aluminum adjuvant.
  • the VLPs of at least one type of HPV include an HPV type selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82.
  • HPV type selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82.
  • the pharmaceutical compositions include VLPs of HPV types 6, 11, 16, and 18.
  • compositions comprise VLPs of HPV types 6, 11, 16, 18, 31, 45, 52, and 58. In further embodiments, the compositions comprise VLPs of HPV types 6, 11, 16, 18, 31, 35, 45, and 58. In still further embodiments, the compositions of the invention, including the quadrivalent and octavalent compositions described, supra, further comprise VLPs of at least one additional HPV type, wherein the at least one additional HPV type is selected from the group consisting of: HPV26, HPV33, HPV35, HPV39, HPV51, HPV53, HPV55, HPV56, HPV59, HPV66, HPV68, HPV73, and HPV82.
  • compositions of this aspect of the present invention comprise an aluminum adjuvant, which effectively enhances the humoral immune response to an HPV type of interest, when VLPs are adsorbed to the aluminum adjuvant and administered to a patient.
  • the aluminum adjuvant is aluminum hydroxyphosphate (AH) or amorphous aluminum hydroxyphosphate sulfate (AAHS). It is preferred that, in these specific embodiments, the aluminum adjuvant comprises phosphate and aluminum present in a molar ratio of about 0.1 to about 1.1 phosphate (PO 4 ) to aluminum (Al). It is more preferred that the aluminum adjuvant comprises phosphate and aluminum present in a molar ratio of about 0.2 to about 0.5 PO 4 /Al. In alternative embodiments of this aspect of the invention, the aluminum adjuvant is aluminum hydroxide.
  • the ISCOM-type adjuvant of the compositions provided herein may be prepared by any one of several known methods for producing ISCOMs known in the art (for review, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25 (1996 )). Well-known methods of production include a centrifugation method and a dialysis method, as described in Barr, supra. ISCOM adjuvants may also be obtained from a commercial source. ISCOM-type vaccines are formulated with Quillaja saponins, cholesterol, and phospholipids such as phosphphatidylcholine, although it has been found that almost any phospholipid is useful in the formation of an ISCOM particle ( see Barr and Mitchell, supra ).
  • the ISCOM-type adjuvant is a so-called ISCOM-matrix adjuvant, such as ISCOMATRIX® (CSL Ltd., Parkville, Australia), which comprises the ISCOPREP® saponin (CSL Ltd., Parkville, Australia) cholesterol, and dipalmitoylphosphatidylcholine.
  • ISCOMATRIX® CSL Ltd., Parkville, Australia
  • ISCOPREP® saponin CSL Ltd., Parkville, Australia
  • dipalmitoylphosphatidylcholine dipalmitoylphosphatidylcholine.
  • compositions of the present invention optimally comprise between 10 and 200 ⁇ g of ISCOM-matrix adjuvant per 0.5 mL dose of VLP vaccine (20 -400 ⁇ g/mL).
  • the ISCOM-type adjuvant is ISCOMATRIX®.
  • HPV vaccine formulations comprising in each dose:
  • a human papillomavirus (HPV) vaccine formulation comprising: (a) HPV virus-like particles (VLPs) of at least one HPV type, wherein the HPV type is selected from the group consisting of: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73, and HPV82; said HPV VLPs comprised of recombinant L1 or recombinant L1 + L2 protein of HPV; wherein the VLPs of any one HPV type are from 10 ⁇ g to 100 ⁇ g and wherein the total VLP up to 600 ⁇ g; (b) from about 150 ⁇ g to about 600 ⁇ g of aluminum adjuvant; and (c) from about 10
  • the HPV vaccine formulations comprise (a) from 20 ⁇ g to 80 ⁇ g of VLPs of at least one HPV type; (b) from 200 ⁇ g to 300 ⁇ g of the aluminum adjuvant; and (c) from 15 ⁇ g to 120 ⁇ g of the ISCOM-type adjuvant.
  • the formulation further comprises from 20 ⁇ g to 60 ⁇ g of VLPs of at least one additional HPV type.
  • the instant invention relates, in one embodiment, to HPV vaccine formulations comprising VLPs of HPV types 6, 11, 16, and 18.
  • the present invention also provides HPV vaccine formulations, further comprising VLPs of HPV types 31, 45, 52, and 58.
  • the present invention further provides HPV vaccine formulations, as described above, further comprising HPV types 31, 35, 45, and 58.
  • HPV vaccine formulations comprising an ISCOM-type adjuvant, wherein the ISCOM-type adjuvant is ISCOMATRIX®.
  • an HPV vaccine formulation comprising in each dose (a) from 20 ⁇ g to 40 ⁇ g of VLPs of each of HPV types 6, 11, 16, 18, 31, 45, 52, and 58, said HPV VLPs comprised of recombinant L1 or recombinant L1 + L2 protein of HPV; (b) from 200 ⁇ g to 300 ⁇ g of aluminum adjuvant; and (c) from 30 ⁇ g to 120 ⁇ g of an ISCOM-type adjuvant, said ISCOM-type adjuvant comprising a saponin, cholesterol, and a phospholipid; wherein said HPV VLPs are adsorbed onto said aluminum adjuvant.
  • HPV types to be used in the vaccine formulation above may be substituted with other HPV types, or combination thereof, such as an octavalent vaccine formulation comprising HPV types 6, 11, 16, 18, 31, 35, 45, and 58.
  • compositions and formulations of the current invention may be used to induce a durable and efficacious immune response to HPV in a patient, and to prevent HPV infection.
  • the pharmaceutical composition that is to be administered to the patient comprises VLPs of HPV types 6, 11, 16, and 18.
  • the compositions further comprise VLPs of HPV types 31, 45, 52, and 58.
  • the compositions further comprise VLPs of HPV types 31, 35, 45, and 58.
  • the compositions further comprise VLPs of at least one additional HPV type selected from the group consisting of: HPV26, HPV33, HPV35, HPV39, HPV51, HPV53, HPV55, HPV56, HPV59, HPV66, HPV68, HPV73, and HPV82.
  • Vaccine compositions of the present invention may be used alone at appropriate dosages which allow for optimal inhibition of HPV infection with minimal potential toxicity.
  • co-administration or sequential administration of other agents may be desirable.
  • compositions of the present invention may be administered to a patient by intramuscular injection, subcutaneous injection, intradermal introduction, or impression though the skin.
  • Other modes of administration such as intraperitoneal, intravenous, or inhalation delivery are also contemplated.
  • the vaccines and pharmaceutical compositions are administered by intramuscular administration.
  • HPV pharmaceutical compositions and formulations disclosed herein may be administered to a patient in various prime/boost combinations in order to induce an enhanced, durable, immune response.
  • two pharmaceutical compositions are administered in a "prime and boost" regimen.
  • the first composition is administered one or more times, then after a predetermined amount of time, for example, 2 weeks, 1 month, 2 months, six months, or other appropriate interval, a second composition is administered one or more times.
  • the two or more HPV pharmaceutical compositions used in a clinical regimen comprise VLPs of the same HPV type or combination of HPV types.
  • a vaccine composition comprising HPV 16 and 18 VLPs may be administered at one point in time, followed by an HPV vaccine composition comprising HPV 31, 45, 52, and 58 VLPs at a second point in time, after a pre-determined length of time has passed.
  • each of the two different HPV vaccine compositions may be administered to the patient once, or more than one time, separated by an appropriate length of time.
  • Also described is a method of preventing HPV infection in a human patient comprising: (a) introducing into the patient a first pharmaceutical composition comprising recombinant HPV VLPs, aluminum adjuvant, and an ISCOM-type adjuvant to the patient, wherein said ISCOM-type adjuvant comprises a saponin, a sterol, and a phospholipid, wherein the HPV VLPs are comprised of recombinant L1 protein or recombinant L1 + L2 protein of HPV and wherein said VLPs are adsorbed to the aluminum adjuvant; (b) allowing a predetermined amount of time to pass; and (c) introducing into the patient a second pharmaceutical composition comprising recombinant HPV VLPs, aluminum adjuvant, and an ISCOM-type adjuvant to the patient, wherein said ISCOM-type adjuvant comprises a saponin, a sterol, and a phospholipid, wherein the HPV VLPs are comprised of recombinant
  • the first and second compositions may be the same and the clinical regimen includes at least one injection of the composition to "prime” the immune response to HPV and at least one injection to "boost” the immune response.
  • the clinical regimen includes at least one injection of the composition to "prime” the immune response to HPV and at least one injection to "boost” the immune response.
  • multiple injections to prime and/or boost the immune response are also contemplated.
  • multi-dose HPV vaccine formulation which comprises more than one dose of vaccine in the same vial.
  • an anti-microbial preservative should be used to kill or prevent the growth of microorganisms, such as bacteria and fungi.
  • Multi-dose vaccine formulations containing anti-microbial preservatives provide several advantages over single dose formulations, including allowing multiple doses of vaccine to be withdrawn from the vial over a period of time without the concern that the first withdrawal inadvertently introduced microbial contamination ( Meyer et al., J. Pharm. Sci. 96(12): 3155-3167 (2007 )).
  • antimicrobial preservatives selected from the group consisting of: m-cresol, phenol, and benzyl alcohol are effective at reducing or eliminating microbes and does not negatively impact the structural and thermal stability of the VLPs at 2-8°C. It is also shown herein that anti-microbial preservatives selected from the group consisting of: m-cresol, phenol, and benzyl alcohol are an effective preservative in HPV vaccine formulations comprising HPV VLPs in combination with an ISCOM-type adjuvant.
  • HPV vaccine formulations comprising HPV VLPs and an antimicrobial preservative selected from the group consisting of: m-cresol, phenol and benzyl alcohol may also include and ISCOM-type adjuvant and an aluminum adjuvant, as described above.
  • m-cresol is included in the multi-dose HPV vaccine formulation at a concentration of about 0.15 to about 0.31%. In more preferred embodiments, the multi-dose vaccine formulations comprise m-cresol at a concentration of about 0.25 to about 0.31 %. In one preferred embodiment, m-cresol is included in the multi-dose formulation at a concentration of about 0.3%.
  • phenol is included in the multi-dose HPV VLP vaccine formulations at a concentration of about 0.25 to about 0.55%. In more preferred embodiments, phenol is included at a concentration of about 0.4 to about 0.55%. In one particularly preferred embodiment, a multi-dose HPV VLP vaccine formulation comprising phenol at a concentration of about 0.5% is provided.
  • the multi-dose HPV vaccine formulations comprise benzyl alcohol at a concentration of about 0.75 to about 1.2%. In more preferred embodiments, benzyl alcohol is included in the multi-dose vaccine formulation at a concentration from about 0.8% to about 1.0%. In a particularly preferred embodiment of this aspect of the invention, the concentration of benzyl alcohol is 0.9%.
  • the multi-dose HPV vaccine formulation described above may optionally include an ISCOM-type adjuvant
  • an octavalent HPV VLP vaccine was administered to rhesus macaques in combination with amorphous aluminum hydroxyphosphate sulfate (AAHS) plus the ISCOMATRIX® adjuvant
  • the vaccine consisted of eight different HPV L1 virus-like particles.
  • the VLPs were adsorbed onto aluminum hydroxide phosphate adjuvant (MAA) present at 450 mcg/mL concentration.
  • MAA-adsorbed vaccines were tested either with or without further addition of ISCOMATRIX® (CSL Limited ABN, Parkville, Australia) present at 200 mcg/mL.
  • the vaccines were in buffer that consisted of 0.32 M NaCl, 10 mM Histidine (pH 6.2) and 0.01% polysorbate-80. A single injection of 0.5-mL volume of the vaccine was given to the right deltoid of each Indian rhesus macaque. Table 1.
  • Results indicate that all animals that were immunized with either the MAA-adjuvanted or the MAA/IMX-adjuvanted vaccine seroconverted to each of the eight HPV types.
  • the geometric mean titers against each of the HPV types were consistently elevated in the MAA/IMX-adjuvanted vaccine recipients (Group II) compared to the MAA-adjuvanted vaccine recipients (Group I).
  • the improvement remained apparent at week 52, suggesting that the effect is long-lived.
  • cLIA competitive Luminex immunoassay
  • HPV cLIA utilizes eight type-specific, conformational dependent mAbs to monitor the immune response to immunization with an HPV Octavalent vaccine.
  • a more detailed description of the competitive Luminex immunoassay which utilizes particle-based flow cytometric analysis to simultaneously measure the presence of antibodies to a plurality of HPV types in a test sample, is set forth in U.S. Patent No. 7,067,258 , and in Opalka et al. Clin Diagn Lab Immunol. 10(1):108-15 (2003 ).
  • An octavalent reference standard solution was prepared for each HPV type in a pre-screened assay matrix containing Antibody Depleted Human Serum (ADHS).
  • the reference standard was a pool of serum samples from Rhesus macaques hyper-immunized with HPV monovalent L1 VLP types 6, 11, 16, 18, 31, 45, 52, 58. All standards, controls and samples were tested in duplicate. Testing of serum samples were performed at a 1:4, 1:40, 1:400, or 1:4000 dilution. Fluorescent units were read and duplicate wells averaged. Dilution corrected mMU/mL serum values were computed based on four parameter logistics fit of the standard curve on each assay plate.
  • ISCOMATRIX® ISCOMATRIX®
  • HPV VLP vaccine adjuvanted with amorphous aluminum hydroxyphosphate sulfate (MAA) Subjects were randomized in two separate study phases.
  • IMX concentrations of 15 or 30 mcg (Phase A) and 60 or 120 mcg (Phase B) per each 0.5-mL dose were evaluated in the context of an experimental octavalent vaccine containing a fixed concentration of HPV VLPs formulated with MAA (281 mcg) and compared to GARDASIL®.
  • the octavalent vaccine contained HPV VLPs of types 6 (20 mcg), 11 (40 mcg), 16 (40 mcg), 18 (20 mcg), 31 (20 mcg), 45 (20 mcg), 5 (20 mcg), and 58(20 mcg).
  • GARDASIL® is a quadrivalent HPV (Types 6, 11, 16, 18) recombinant vaccine prepared from highly purified L1 VLPs.
  • each vaccine dose contained MAA (225 mcg), sodium chloride, L-histidine, polysorbate 80, sodium borate, and water.
  • vaccine was administered at day 1, month 2, and month 6 as a 0.5-mL intramuscular injection of either one of the four formulations of octavalent HPV L1 VLP with MAA/IMX described above, or GARDASIL®.
  • the study included ⁇ 150 healthy women between the ages of 18 and 24 years at the time of enrollment. Blood samples taken at day 1, month 2, month 3 (1 month post-dose 2), month 6, month 7 (1 month post-dose 3), and month 12 were used for evaluation of immune response to relevant HPV types using a competitive Luminex immunoassay (cLIA), as described in EXAMPLE 2.
  • cLIA competitive Luminex immunoassay
  • Subjects in Phase B were included in the analysis if they: (i) received the first 2 vaccinations in the appropriate day ranges, (ii) had serology samples collected in appropriate day ranges, and (iii) were seronegative to the relevant HPV types at Day 1.
  • results at both the month 3 (postdose 2) and month 7 (postdose 3) time-points by treatment group for subjects in Phase A (15 and 30 mcg IMX, + MAA) and at the month 3 time-point for subjects in Phase B (60 and 120 mcg IMX, + MAA) are displayed in FIGURES 3 and 4 , respectively.
  • the results demonstrate an IMX dose-related enhancement of the immune response to relevant HPV types compared to that of GARDASIL®, which is adjuvanted with MAA alone.
  • HPV VLPs in the following amounts: 40 ⁇ g/mL HPV 6, 80 ⁇ g/mL HPV 11, 80 ⁇ g/mL HPV 16, and 40 ⁇ g/mL each of HPV 18, 31, 45, 52, and 58 VLPs. Also included in each of the formulations was 0.32 M NaCl, 10mM Histidine pH 6.2 and 0.0 1 % polysorbate 80 (PS80).
  • the first formulation contained no IMX or MAA adjuvant.
  • the second formulation contained 240 ⁇ g/mL IMX and no MAA.
  • the third formulation that was tested contained no IMX and 562.5 ⁇ g/mL (1.25X) MAA.
  • the fourth formulation that was tested contained both 240 ⁇ g/mL IMX and 562.5 ⁇ g/mL (1.25X) MAA.
  • Samples were analyzed by SDS-PAGE with Invitrogen 4-20% or 14%, 1.5mm, 10 well Tris-Glycine gels. Samples are prepared in a reducing sample buffer (0.25M Tris PH 6.8, 8% SDS, 40% Glycerol, 0.008% Bromophenol blue, dithiothreitol, and heated at 70°C for 10 minutes). The samples containing aluminum adjuvant were concentrated to 762 ⁇ g/mL prior to sample preparation, and 0.5M EDTA, pH 8.0 was included to dissolve the aluminum.
  • the high load was added to a 1X reducing sample buffer (portions of one 4X reducing sample buffer: three MilliQ water) to prepare a low load (2.5 ⁇ g).
  • a 12 mcg (high) and a 2.5 mcg (low) load were run for each sample.
  • the potencies of HPV types 6, 11, 16, 18, 31, 45, and 52 VLP samples were quantified relative to a reference standard by in vitro relative potency (IVRP) testing.
  • the IVRP assay is a sandwich-type immunoassay that uses separate monoclonal antibodies for capture and detection.
  • the antibodies used in each of the eight serotype-specific assays were prepared by Strategic BioSolutions (Newark, DE).
  • the following serotype-specific capture antibodies were used in the IVRP assay: H6.M48 mAb (IgG 1 ) for HPV6, K11.B2 mAb (IgG 1 ) for HPV11, H16.J4 mAb (IgG 2a ) for HPV16, H18.J4 mAb (IgG 2a ) for HPV18, H31.5F12.D4 mAb (IgG 1 ) for HPV31, H45.6G6.A8 mAb (IgG 2b ) for HPV45, H52.8D11.C4 mAb (IgG 2b ) for HPV52, and H58.2C3.F7 mAb (IgG 1 ) for HPV 58.
  • serotype-specific detection antibodies were used in the assay: H6.B10.5 mAb (IgG 2b ) for HPV6, H11.B2 mAb (IgG 2b ) for HPV11, H16.V5 mAb (IgG 2b ) for HPV16, H18.R5 mAb (IgG 2b ) for HPV18, M31.5D10.E4 mAb (IgG 2b ) for HPV31, H45.10B4.H4 mAb (IgG 1 ) for HPV45, H52.9F7.E10 mAb (IgG 2a ) for HPV52, and H58.6E11.F4 mAb (IgG 2b ) for HPV58.
  • the assay was carried out on a Tecan Freedom EVO workstation (Tecan USA; Research Triangle, NC) using white FluoroNunc MaxiSorp 96-well microtiter plates (Fisher Scientific; Pittsburgh, PA). Assay plates were first coated with the capture antibody (100 ⁇ L/well), in 1 M ammonium sulfate buffered with 10 mM Tris, pH 8, and then allowed to incubate for 16-24 hrs at 2-8 °C.
  • TBS Tris-buffered Saline
  • assay diluent TBS containing 0.05% Tween 20 and 1 % bovine serum albumin
  • adjuvant-adsorbed samples and standards were diluted 5-fold in 6% citrate phosphate buffer (pH 6.7-6.8) containing 1 M NaCl and 0.04% Tween 80 and incubated for 2 + 0.5 hrs at ambient temperature on a nutator for adjuvant dissolution. Samples that were not adsorbed to adjuvant were also diluted in the dissolution buffer but were not incubated.
  • Each sample and reference standard was then diluted to a serotype-specific target concentration with assay diluent in a Nunc 96-well polypropylene microtiter plate from Fisher Scientific (Pittsburgh, PA). From this initial dilution, eleven two-fold serial dilutions were prepared for each sample and for the reference standard in the assay plate. After a 12-20 hour incubation at room temperature with shaking, the plates were washed and 100 ⁇ L of the detection antibody was subsequently added to each well. The plate was incubated for one hour at room temperature and again washed.
  • rat anti-mouse monoclonal antibody (IgG 1 , IgG 2a , or IgG 2b ) conjugated to alkaline-phosphatase was added to each well, supplied from Southern Biotechnology (Birmingham, AL) or BD Pharmingen (BD Biosciences; San Jose, CA). After a one-hour incubation at room temperature, the plate was washed to remove any unbound conjugate. Next, 100 ⁇ L of 4-methylumbelliferyl phosphate (4-MUP) substrate solution from ViroLabs (SuperPhos; Chantilly, VA) was added and allowed to react for one hour.
  • 4-methylumbelliferyl phosphate (4-MUP) substrate solution from ViroLabs (SuperPhos; Chantilly, VA) was added and allowed to react for one hour.
  • the fluorescence of each well was measured by reading the plate in a Tecan Ultra Evolution 384 plate reader (Tecan USA; Research Triangle, NC) at an excitation wavelength of 360 nm and an emission wavelength of 450 nm.
  • IVRP ED 50 ⁇ sample / ED 50 ⁇ standard x Reference Standard Potency
  • the results were subsequently normalized by dividing by the protein concentration, thus producing the IVRP-to-protein ratio.
  • the NRP-to-protein ratio provides a measure of the functional epitopes per unit of mass.
  • AME testing was used to determine the ability of a preservative to reduce or eliminate microbes that may be introduced into the vial following product withdrawal.
  • USP United States Pharmacopoeia
  • AME testing was used to determine the ability of a preservative to reduce or eliminate microbes that may be introduced into the vial following product withdrawal.
  • the AME test comprises the EPA, EPB and USP criteria, which consist of inoculating 10 5 -10 6 CFU/mL microorganisms (e.g., bacteria and fungi) per container at time zero, and evaluating the log reduction over time (Meyer et al., supra ).
  • CFU/mL microorganisms e.g., bacteria and fungi
  • the initial preservative screening was conducted using the following seven preservatives: benzyl alcohol, chlorobutanol (plus EtOH), m-cresol, methylparaben, propylparaben, phenol, and 2-phenoxyethanol in various doses and combinations. These preservatives have been used as anti-microbial preservatives in commercial parenteral products. Thimerosal was also included as a reference.
  • the initial doses and combinations of the preservatives were selected based on previous work and available literature. More specifically, concentrations were tested ranging from 0.5-1.2% benzyl alcohol; 0.25-0.5% chlorobutanol (0.5% EtOH or 3.0% EtOH); 0.15-0.3% m-cresol; 0.045-0.18% methylparaben; 0.005-0.02% propylparaben; 0.25-0.5% phenol; 0.25-0.5% 2-phenoxyethanol; and 0.002-0.01% thimerosal. All samples contained 562.5 mcg/mL MAA, 0.32 M NaCl, 10 mM His, 0.01% polysorbate-80 (PS-80), pH 6.2. Formulations containing chlorobutanol and propylparaben were tested with and without the addition of 120 mcg/mL ISCOMATRIX®.
  • Benzyl alcohol, phenol, 2-phenoxyethanol, and m-cresol were provided as solutions and were added directly to the HPV formulations.
  • Chlorobutanol was provided in a powder and was dissolved in 100% ethanol to make a stock solution.
  • Thimerosal was dissolved in USP water to make a stock solution.
  • the combination of methylparaben and propylparaben were made by weighing out each powder and making a stock solution in 100% ethanol. The pH of the solution was adjusted with a target pH of 6.2.
  • Preservatives were added to octavalent HPV formulations comprising 8 types of HPV VLPs, as described in EXAMPLE 1, and were stored for one week at 2-8°C prior to AME testing.
  • Formulations containing 1.2% benzyl alcohol, 0.5% chlorobutanol, and 0.3% m-cresol passed the USP, EP-B, and EP-A criteria of the AME test ( See Meyer et al., supra ).
  • Formulations containing 0.4% and 0.5% phenol passed USP and EP-B, but not EP-A tests. Additional preservative-containing formulations were tested that only passed the USP test. Therefore, formulations containing 1.2% benzyl alcohol, 0.5% chlorobutanol, 0.3% m-cresol, and phenol at a concentration of 0.4% or 0.5% were selected for further evaluation.
  • DSC Differential Scanning Calorimetry
  • HPV 18 VLPs were chosen as the model HPV antigen for DSC analysis because this type of VLP gives the most robust DSC signals and represents a less stable type of VLP compared to other types tested.
  • the DSC evaluation was carried out in the presence of various preservatives with varied concentrations. The results indicate that 0.5% chlorobutanol, 0.15% m-cresol, 0.25% phenol, 0.5% ethanol (solvent for chlorobutanol), and 0.25% 2-phenoxyethanol had the least impact on the thermal stability of HPV type 18 (data not shown). The greatest impact on the thermal stability was observed with thimerosal, 1.2 % benzyl alcohol, and 0.045 / 0.005 methylparaben.
  • the RP-HPLC method was also used for evaluating preservative stability in HPV vaccine formulations.
  • a short-term preliminary study showed that the selected preservatives were fairly stable when the HPV vaccine was stored at 2-8°C (data not shown). More detailed evaluations with long-term stability studies are described in EXAMPLE 11-12.
  • Each type of HPV VLP aqueous bulk was absorbed on MAA separately with a protein-to- adjuvant ratio of 0.71 to form the monovalent vaccines in 0.32 M NaCl, 10 mM Histidine (pH 6.2), 0.01% (w/v) PS-80.
  • the eight individual monovalent vaccines were then blended together to obtain protein concentrations of 40, 80, 80, 40, 40, 40, 40 and 40 mcg/mL for HPV Types 6, 11, 16, 18, 31, 45, 52, and 58, respectively. This combination formed the octavalent vaccine.
  • IMX Isocomatrix®
  • HPV vaccine buffer described above.
  • IMX was then added to the octavalent vaccine to a final concentration of 240 mcg/mL using a settle-decant procedure to form the octavalent HPV vaccine containing IMX adjuvant.
  • the formulations were gently mixed and stored at 4°C overnight.
  • the initial preservative screening (EXAMPLE 7) used 120 mcg/mL IMX in the chlorobutanol formulation because the final IMX concentration had not been determined when these experiments were performed.
  • the formulations were filled at a volume of 0.75 mL in 3 mL glass vials with Teflon-coated stoppers (FluroTec®, West Pharmaceuticals, Lionville, PA) and stored in controlled thermal units at 2-8, 25, 30, and 37°C prior to testing.
  • Teflon-coated stoppers FluroTec®, West Pharmaceuticals, Lionville, PA
  • the formulation and fill processes were performed under aseptic conditions.
  • the measurements are scheduled to be at 0, 8, 12, 24, and 36 months for preservative, HPV VLP, and IMX adjuvant testing.
  • Formulations from the real-time study (2-8°C) were first evaluated at time 0 for preservative effectiveness using AME testing.
  • the formulations containing 0.5% chlorobutanol and 0.3% m-cresol in the presence or absence of IMX passed the USP, EP-B and EP-A tests.
  • the formulation containing 1.2% benzyl alcohol without IMX passed USP, EP-B, and EP-A, but did not pass EP-A in the IMX-containing formulation.
  • Formulations that did not pass EP-A in the presence or absence of IMX also included 0.9% benzyl alcohol and 0.5% phenol. 3.3.3. Stability Data for 8 Months Storage at 2-8 °C and 3 Months at 30 and 37 °C

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Claims (14)

  1. Eine pharmazeutische Zusammensetzung, umfassend in jeder Dosis:
    (a) 10 bis 100 µg pro Typ virusartige Partikel (VLPs) von wenigstens einem Typ von humanem Papillomavirus (HPV),
    (b) 300 bis 1200 µg/ml eines Aluminium-Hilfsstoffes,
    (c) 20-400 µg/ml eines Hilfsstoffes vom ISCOM-Typ, umfassend einen immunstimulierenden Komplex, der ein Saponin, Cholesterin und ein Phospholipid beinhaltet, und
    (d) einen pharmazeutisch annehmbaren Träger,
    wobei die VLPs ein rekombinantes L1-Protein oder rekombinante L1+L2-Proteine von HPV beinhalten, und wobei die VLPs auf dem Aluminium-Hilfsstoff adsorbiert sind.
  2. Die pharmazeutische Zusammensetzung nach Anspruch 1, wobei der wenigstens eine Typ HPV-VLPs ausgewählt ist aus der Gruppe, bestehend aus: HPV6, HPV11, HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV55, HPV56, HPV58, HPV59, HPV66, HPV68, HPV73 und HPV82.
  3. Eine pharmazeutische Zusammensetzung nach Anspruch 2, umfassend in jeder Dosis:
    (a) bis zu 600 µg VLPs insgesamt,
    (b) 150 µg bis 600 µg Aluminium-Hilfsstoff und
    (c) 10 µg bis 200 µg eines Hilfsstoffes vom ISCOM-Typ,
    wobei die HPV-VLPs auf dem Aluminium-Hilfsstoff adsorbiert sind.
  4. Die pharmazeutische Zusammensetzung nach Anspruch 3, ferner umfassend ein antimikrobielles Konservierungsmittel, ausgewählt aus der Gruppe, bestehend aus: m-Cresol, Phenol und Benzylalkohol.
  5. Die pharmazeutische Zusammensetzung nach Anspruch 3, umfassend in jeder Dosis:
    (a) 20 µg bis 80 µg VLPs jeweils pro HPV-Typ,
    (b) 200 µg bis 300 µg des Aluminium-Hilfsstoffes und
    (c) 15 µg bis 120 µg des Hilfsstoffes vom ISCOM-Typ,
    oder
    (a) 20 µg bis 80 µg VLPs jeweils pro HPV-Typ,
    (b) 300 µg bis 500 µg des Aluminium-Hilfsstoffes und
    (c) 15 µg bis 120 µg des Hilfsstoffes vom ISCOM-Typ.
  6. Die pharmazeutische Zusammensetzung gemäß einem der Ansprüche 1, 2 und 3, umfassend VLPs der HPV-Typen 6, 11, 16 und 18.
  7. Die pharmazeutische Zusammensetzung nach einem der Ansprüche 1-3 und 6, umfassend VLPs der HPV-Typen 31, 45, 52 und 58.
  8. Die pharmazeutische Zusammensetzung gemäß einem der Ansprüche 2, 6 und 7, ferner umfassend VLPs eines zusätzlichen HPV-Typs, ausgewählt aus der Gruppe, bestehend aus:
    HPV26, HPV33, HPV35, HPV39, HPV51, HPV53, HPV55, HPV56, HPV59, HPV66, HPV68, HPV73 und HPV82.
  9. Die pharmazeutische Zusammensetzung nach einem der Ansprüche 1-2 und 6-8, wobei der Aluminium-Hilfsstoff ausgewählt ist aus der Gruppe, bestehend aus:
    Aluminiumhydroxyphosphat (AlPO4), amorphem Aluminiumhydroxyphosphatsulfat (AAHS) und Aluminiumhydroxid (AI(OH)3).
  10. Die pharmazeutische Zusammensetzung nach Anspruch 9, wobei der Aluminium-Hilfsstoff AlPO4 oder AAHS umfasst, wobei der Hilfsstoff Phosphat und Aluminium umfasst, die in einem Molverhältnis von 0,1 bis 1,1 Phosphat (PO4) zu Aluminium (Al) vorliegen.
  11. Die pharmazeutische Zusammensetzung nach Anspruch 9 oder 10, wobei der Aluminium-Hilfsstoff Phosphat und Aluminium umfasst, die in einem Molverhältnis von 0,2 bis 0,5 PO4/Al vorliegen.
  12. Die pharmazeutische Zusammensetzung gemäß einem der Ansprüche 1-3 und 6-11, wobei der Hilfsstoff vom ISCOM-Typ ISCOMATRIX® ist.
  13. Die pharmazeutische Zusammensetzung nach Anspruch 3, umfassend in jeder Dosis:
    (a) jeweils 20 µg bis 80 µg VLPs der HPV-Typen 6, 11, 16, 18, 31, 45, 52 und 58, wobei die HPV-VLPs rekombinantes L1- oder rekombinantes L1+L2-Protein von HPV beinhalten,
    (b) 200 µg bis 500 µg Aluminium-Hilfsstoff und
    (c) 30 µg bis 120 µg eines Hilfsstoffes vom ISCOM-Typ,
    wobei die HPV-VLPs auf dem Aluminium-Hilfsstoff adsorbiert sind.
  14. Die pharmazeutische Zusammensetzung nach Anspruch 4, wobei das antimikrobielle Konservierungsmittel aus m-Cresol in einer Konzentration von 0,15 bis 0,31%, Phenol in einer Konzentration von 0,25 bis 0,55% oder Benzylalkohol in einer Konzentration von 0,75 bis 1,2% besteht.
EP08726508A 2007-03-09 2008-03-06 Impfstoffzusammensetzung gegen papillomavirus Not-in-force EP2129394B1 (de)

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EP2129394A1 (de) 2009-12-09
US20120177684A1 (en) 2012-07-12
JP5956974B2 (ja) 2016-07-27
CA2679720A1 (en) 2008-09-18
US20080248062A1 (en) 2008-10-09
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CN101622008A (zh) 2010-01-06
WO2008112125A1 (en) 2008-09-18
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